Parasite infection is responsible for a number of human and animal diseases.
Parasitic protozoa of the order Kinetoplastidae are the causative agents of several tropical diseases including sleeping sickness by Trypanosoma brucei, Chagas by Trypanosoma cruzi, visceral (kala-azar) and cutaneous (oriental sore) Leishmaniasis by Leishmania donovani and Leishmania major, respectively. In particular Leishmania protozoans are the causative agents of human leishmaniasis, which includes a spectrum of diseases ranging from self-healing skin ulcers to fatal visceral infections. Human leishmaniasis is caused by at least thirteen different species and subspecies of parasites of the genus Leishmania. Leishmaniasis has been reported from about eighty countries and probably some 400,000 new cases occur each year. Recently the World Health Organization has reported 12 million people to be infected (ref. 1. Throughout this application various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosure of these references are hereby incorporated by reference into the present disclosure. A listing of the references appears at the end of the disclosure).
Untreated visceral leishmaniasis is usually fatal and mucocutaneous leishmaniasis produces mutilation by destruction of the naso-oropharyngeal cavity and, in some cases, death.
In addition a major health problem has been created in areas of high infection when blood is collected for transfusion purposes. Since blood is a carrier of the parasites, blood from an infected individual may be unknowingly transferred to a healthy individual.
The Leishmania protozoans exist as extracellular flagellated promastigotes in the alimentary tract of the sandfly in the free-living state, and are transmitted to the mammalian host through the bite of the insect vector. Once introduced, the promastigotes are taken up by macrophages, rapidly differentiate into non-flagellated amastigotes and start to multiply within the phagolysosomal compartment. As the infected cells rupture, amastigotes subsequently infect other macrophages giving rise to the various symptoms associated with leishmaniasis (refs. 1 and 2).
Leishmaniasis is, therefore, a serious disease and various types of vaccines against the disease have been developed, including live parasites; frozen promastigotes from culture; sonicated promastigotes; gamma-irradiated live promastigotes; and formalin-killed promastigotes treated with glucan (reviewed in, for example ref. 3). However, none of these approaches have provided efficacious vaccines.
Healing and progression of leishmaniasis are linked to the dissimilar expansion of functionally distinct CD4+ lymphocyte responses separated on the basis of their cytokine potential (ref. 4). T helper type 1 (Th1) subset produces interferon (IFN)-,γ and interleukin (IL)-2 and leads to resistance to Leishmania infection, whereas Th2 cells producing IL-4, IL-5 and IL-10 confer susceptibility (ref. 5). In mammalian hosts, Leishmania reside exclusively within mononuclear phagocytes, macrophages and monocytes. Cytokines can modulate macrophage differentiation by causing selective changes in macrophage gene expression, leading to alterations on macrophage functions (ref. 6). Macrophages, pre-incubated in vitro with cytokines prior to infection with Leishmania, acquire the capacity to kill the intracellular parasites (refs. 7 to 11). Furthermore, cytokines such as IFN-γ, TNF-α, IL-12 and GM-CSF have been used in anti-leishmanial therapy in experimental models (refs. 12 to 17). Expression of the IFN-γ gene has been performed in L. major trypanosomatids. When nude mice were infected with the IFN-γ expressing transfectant, the progression of the disease was considerably slower (ref. 17). The progression of the disease was not retarded in susceptible BALB/c mice however.
GM-CSF is a cytokine with multipontential hematopoietic function, stimulating the formation of granulocytes, macrophages, and eosinophils (ref. 18). It activates macrophage tumoricidal activity (ref. 19), increases macrophage killing of Trypanosoma cruzi (ref. 20) and enhances in vitro killing of L. donovani within macrophages (ref. 8).
Differences on the effect of this cytokine have been reported between experimental visceral and cutaneous infections (ref. 27). Although in the L. donovani model, GM-CSF demonstrates a clear-cut leishmanicidal activity in vitro and in vivo (refs. 28), the results obtained with L. major are conflicting suggesting that GM-CSF may play a positive (refs. 29, 10, 6), neutral (refs. 30, 4) or negative (ref. 31) host defense role.
Parasitic infection of macrophages and, in particular, Leishmania infection may lead to serious disease. It would be advantageous to provide attenuated strains of Leishmania and methods of production thereof, for use as antigens in immunogenic preparations, including vaccines, and the generation of diagnostic reagents.